In many industries it is desirable or necessary to regularly monitor the concentration of particular constituents in a fluid. A number of systems are available that analyze the constituents of bodily fluids such as blood, urine, or saliva. Examples of such systems conveniently monitor the level of particular medically significant fluid constituents, such as, for example, cholesterol, ketones, vitamins, proteins, and various metabolites or blood sugars, such as glucose. Diagnosis and management of patients suffering from diabetes mellitus, a disorder of the pancreas where insufficient production of insulin prevents normal regulation of blood sugar levels, requires carefully monitoring of blood glucose levels on a daily basis. A number of systems that allow individuals to easily monitor their blood glucose are currently available. Such systems include electrochemical biosensors, which may comprise a test strip wherein a user applies a blood sample and a meter “reads” the test strip by correlating the current detected with glucose concentration in the blood sample.
Among the various technologies available for measuring bodily fluid constituents such as blood glucose, electrochemical technologies are particularly useful because a very small blood sample (in some cases as low as 300 nL) may suffice to perform the measurement. In amperometric electrochemical-based systems, the test strip is typically imprinted or screen-printed with working and reference electrodes. The working electrode may be coated or deposited with reagents such as enzymes that catalyze oxidation/reduction (“redox”) reactions, enzyme mediators and membranes. A counter or “non-working” electrode is included, along with an optional additional “reference” electrode, also known as a “baseline” electrode. The reference electrode may be described as “non-working.”
The measurement of medically relevant bodily fluid constituents may include one or more of the following enzyme reagents: glucose oxidase, glucose dehydrogenase, cholesterol esterase, cholesterol oxidase, lipoprotein lipase, glycerol kinase, glycerol-3-phosphate oxidase, lactate oxidase, lactate dehydrogenase, pyruvate oxidase, alcohol oxidase or bilirubin oxidase. In the case of blood glucose measurements, a user typically applies a blood sample to the sample chamber, an enzyme such as glucose oxidase reacts with the glucose, and a voltage is applied across electrode one working and one non-working electrode in the sample chamber, thereby causing a redox reaction to occur. A meter then measures the resulting current and relates this to the amount of glucose in the original sample. Other systems based on coulometry or voltametry are also known. A common feature of all of these systems is the use of standard or control solutions with a known concentration of glucose to verify that the measurement apparatus is operating correctly and to ensure the accuracy of diagnostic tests.
Current technology typically requires a manual determination of whether the sample is of a control solution or a bodily fluid, such as blood. This can be problematic for several reasons; in particular, a patient's poor eyesight or lack of dexterity can make a manual selection to indicate whether the solution is a sample or control solution quite difficult. An error in this manual entry will result in an erroneous average, which can significantly affect a patient's choice of treatment options. For many patients, such manual intervention presents a substantial physical challenge. A distinct issue unrelated to the physical challenge of making such a manual adjustment, is that individuals who are responsible for showing their average glucose (a current function of most monitors) might willfully adjust their average glucose readings by using the low or normal glucose level control solution, and in this way lower, their average glucose level. Such a situation may be encountered when an individual's own actions would render the actual average glucose levels higher than desired. To further illustrate this example, when a teenager consumes food or beverages that violate a strict diet plan, he or she might be able to falsify the average glucose reading by substituting the blood sample with the low or normal glucose level control solution. Thus, manual determination of a control solution remains a substantial problem in patient care.
As stated above, electrochemical biosensor technologies typically make use of some electrode based biosensors, and a number of configurations are commonly used to support accurate measurements and meet design constraints. Specifically, such biosensors typically include at least one working electrode and at least one counter electrode, which may be on the same substrate (e.g., co-planar) or may be on different substrates (e.g., facing). Such biosensors also typically include a sample chamber to hold the sample in electrolytic contact with the working electrode. Common configurations include at least one working electrode on a first substrate and forming at least one counter or reference electrode on a second substrate. A spacer layer is disposed on either the first or second substrates. The spacer layer defines a chamber into which a sample may be drawn and held when the biosensor is completed. Chemical detection of one or more analytes may be configured on the first or second substrate in a region that will be exposed within the sample chamber when the biosensor is completed. The first and second substrates may then be brought together as a “sandwich” and spaced apart by the spacer layer with the sample chamber providing access to the at least one working electrode and the at least one counter or reference electrode.
Certain other embodiments include forming at least one working electrode on a first substrate and forming at least one counter or reference electrode on the same, first substrate. One or two additional layers may be added to define a chamber into which a sample may be drawn and held when the biosensor is completed. A “trigger” electrode may also be included to indicate when the chamber has filled with sample solution.
As provided above, the biosensor may include a working electrode and at least one counter electrode. The working electrode is the electrode through which electrons from glucose enter the biosensor. The counter electrode is where the electrons exit the sensor and return to the fluid sample. Although two electrodes is a minimum number for any electrochemical sensing device, more than two electrodes can be used. For example, a third reference, or baseline electrode may be included, which severs as a reference point to precisely set the potential (or potential to accept electrons) of the working electrode. The reference electrode may be considered a “non-working” electrode. Biosensors of the invention may include at least two non-working electrodes. In this way, biosensors of the invention can be configured to measure electron conductivity in fluid, as opposed to, or in addition to, glucose concentration. Biosensors configured to measure electron conductivity may comprise a working electrode that is temporarily disconnected or disabled. Disablement of the working electrode allows for the measurement of electron conductivity alone.
Working electrodes may be manufactured from any number of useful materials, typically made up of any combination of one or more conductive materials having in some measure desirable properties of low electrical resistance and electrochemical inertness over the potential range of the biosensor during operation. Gold, carbon, platinum, ruthenium dioxide, palladium, or other non-corroding materials are cited as particularly exemplary working electrode materials. Counter electrodes may be constructed in a manner similar to working electrode. Suitable materials for the counter/reference or reference electrode include Ag/AgCl or Ag/AgBr on a non-conducting base material or silver chloride on a silver metal base, often including a mix of multiple conducting materials, such as Ag/AgCl and carbon.
Various embodiments of methods of making biosensors include providing a sample chamber and/or measurement zone having an electrode surface area that, when filled with a sample to be tested, provides a clinically accurate analyte level reading, preferably without user intervention. In particular, the configurations disclosed here are directed toward a design that minimizes or entirely removes the need for users to manually identify control solutions or experimental solutions such as a blood sample.
Technology does exist for more automated sample discrimination, but such methods have so far been limited to situations in which the sample is blood. Such methods are problematic when the sample is not blood, and is instead urine, interstitial fluid or plasma, for example. Tokunaga et al. (U.S. Pat. Nos. 6,824,670 and 7,122,111) discloses a method of automatic sample discrimination using a discrimination function and index to distinguish a control solution from a sample solution. Importantly, this method uses the electrochemical test electrode, or working electrode, to measure current in the control and sample solutions. This technology is based on an algorithm that identifies differences in the time differential of the current measured in the control and the sample solutions. Essentially, the algorithm identifies changes in the shape of the measurement curve over time as characteristic of control or sample solutions. Such methods, however, are not entirely reliable when the sample measured is not blood.
In addition, these methods require significantly more complex measurement devices that are more expensive to develop and maintain, and the actual determination typically takes an undesirably long period of time to perform. The time differential calculations necessary to distinguish sample from control are on the order of at least several seconds. Such a long waiting period is a substantial disadvantage for users that demand glucose monitoring devices with a premium on convenience. Further, while the methods of the prior art involve a single measurement, the instantly claimed subject matter involves two measurements. The first assesses glucose concentration while the second assesses another parameter, such as, for example, electron conductivity in fluid. Embodiments of the present invention allow both of these measurements to be assessed in less time than the single measurement step of the prior art.
For example, Chatelier et al. (U.S. Application Publication No. 20070235347) describes methods for distinguishing control and sample solutions using a series of current transient measurements that enable discrimination via the measurement of the impact of endogenous redox species in blood termed interferents. This disclosure, however, does not focus on embodiments in which there is more than one non-working electrode. Nor does it describe sequential measurements in which a voltage is applied across the working electrode and a non-working electrode, followed or preceded by application of a voltage across two non-working electrodes. Provided herein is the step-wise application of a voltage across different electrode configurations resulting in two separate measurements. The magnitude of the voltage applied across the working and non-working electrode is typically low, being between about 100-300 mV. This is due to the fact that a glucose signal is relatively easy to detect. In contrast, the magnitude of the voltage applied across non-working electrodes is typically high, between about 300-500 mV. This is due to the fact that measurement of bulk solution conductivity requires a potential of relatively greater magnitude for detection.
Accordingly, the presently claimed subject matter involves applying a voltage between a working and a non-working electrode in order to quantitatively measure analyte concentration, and applying a different voltage between two non-working electrodes in order to measure another parameter, such as, for example electron conductivity. These separate measurements need not occur in any particular order.
It should be noted that the presence of a working electrode results in measurement of analyte concentration, which may interfere with the control solution reading. Thus, in one embodiment, the subject method involves “deactivating” the glucose signal so that the measured signal of the control solution is glucose-independent. This may be accomplished by temporarily disconnecting the working electrode.
It should be emphasized that accurate measurements of concentration levels in a bodily fluid, such as blood, may be critical to the long-term health of many users. As a result, there is a need for a high level of reliability in the meters and test strips used to repeatedly measure concentration levels in fluids. It is therefore desirable to have a cost effective auto-calibration system for diagnostic test strips that more reliably and more accurately provides an average glucose reading. For the same reason, it would be highly beneficial for electrochemical biosensors, and glucose monitoring systems in particular, to accurately, automatically and rapidly distinguish between control and sample solutions.